The invention provides animal models for the evaluation of candidate drugs and therapies for the prevention and treatment of amyloidoses, including Alzheimer""s disease. For example, a rapid animal model of congophilic and fibrillar xcex2/A4 amyloid deposition in brain is provided for in vivo screening of candidate therapeutic agents and protocols. Heparin, heparan sulphate glycosaminoglycans, and related macromolecules, as well as heparin-binding peptides, are provided as therapeutic agents for amyloid deposition in Alzheimer""s disease brain and other amyloidoses.
Amyloid and Amyloidosis
Amyloid is a generic term referring to a group of diverse, but specific extracellular protein deposits which all have common morphological properties, staining characteristics and x-ray diffraction spectra. Regardless of the nature of the amyloid protein deposited all amyloids have the following characteristics: 1) an amorphous appearance at the light microscopic level and appear eosinophilic using hematoxylin and eosin stains; 2) all stain with congo red and demonstrate a red/green birefringence as viewed under polarized light; 3) all contain a predominant beta-pleated sheet secondary structure; and 4) ultrastructurally amyloid usually consists of non-branching fibrils of indefinite length and with a diameter of 8-12 nm.
Amyloidosis: Early Historical Perspectives
Rokitansky in 1842 was the first to observe waxy, eosinophilic tissue deposits in a number of tissues from different patients. However, it wasn""t until 1854 when Virchow termed these deposits as xe2x80x9camyloidxe2x80x9d meaning xe2x80x9cstarch-likexe2x80x9d since they gave a positive staining with the sulfuric acid-iodine reaction, which was used in the 1850s for demonstrating cellulose. Although cellulose is not a constituent of amyloid, nonetheless, the staining that Virchow observed is probably due to the presence of different carbohydrates, known as highly sulfated glycosaminoglycans and proteoglycans, which appear to be associated with all types of amyloid deposits (see below). The name amyloid has remained despite the fact that Freiderich and Kekule in 1859 discovered the protein nature of amyloid.
Amyloid is not a Single Disease
For many years, based on the fact that all amyloids have the same staining and structural properties, lead to the postulate that a single pathogenetic mechanism was involved in amyloid deposition, and that amyloid deposits were thought to be composed of a single set of constituents. Current research has clearly shown that amyloid is not a uniform deposit and that amyloids may consist of different protein which are totally unrelated.
Amyloidosis is not an xe2x80x9cImmune Disorderxe2x80x9d
It is interesting that the pathology textbook by Robbins has xe2x80x9camyloidosisxe2x80x9d under the heading of xe2x80x9cpossible immune disordersxe2x80x9d. Obviously these authors thought like many of the predecessors studying amyloidosis in the 1960s and 1970s. This is based on a number of early observations made about amyloidosis. 1) Clinically, many types of amyloid were due to a complication of long-standing inflammatory disorders such as rheumatoid arthritis or osteomyelitis. 2) Histologically, tissue reactions in many of these disorders were characterized by the presence of immunologically competent cells (e.g. monocytes, macrophages, plasma cells. 3) Amyloid was developed in animals used for raising antisera. Repeated injections of antigens were not uncommonly followed by systemic amyloid deposits. Therefore, clinical, histological, and experimental data focused attention, not illogically, upon the immune system. However, by the mid-late 1970s, the isolation, characterization and sequencing of amyloid proteins from different clinical settings showed that a basic immunological disturbance could not account for the diversity of proteins seen as amyloids in the different diseases.
Clinical Classification of Amyloidosis
Let""s look at how amyloid was classified initially in the mid to late 1970s and then compare it to the classification according to protein type which is used today. Basically, amyloid was clinically classified into 4 groups, primary amyloid, secondary amyloid, familial amyloid, and isolated amyloid.
Primary amyloid, is amyloid appearing de novo, without any preceding disorder. In 25-40% of these cases, primary amyloid was the antecedent of plasma cell dysfunction such as the development of multiple myeloma or other B-cell type malignancies. Here the amyloid appears before rather than after the overt malignancy. Regardless of which clinical element appeared first, the type of amyloid protein in primary amyloid is most often the same as that seen in amyloid secondary to a variety of B-cell dysfunctions.
Secondary amyloid, appears as a complication of a previously existing disorder. 10-15% of patients with multiple myeloma eventually develop amyloid. Patients with rheumatoid arthritis, osteoarthritis, ankylosing spondylitis can develop secondary amyloidosis as with patients with tuberculosis, lung abscesses and osteomyelitis. Intravenous drug users who self-administer and who then develop chronic skin abscesses can also develop secondary amyloid. Secondary amyloid is also seen in patients with specific malignancies such as Hodgkin""s disease and renal cell carcinoma. Although these were all initially classified as secondary amyloid, once the amyloid proteins were isolated and sequenced, many of these turned out to contain different amyloid proteins.
The familial forms of amyloid also show no uniformity in terms of the peptide responsible for the amyloid fibril deposited. Several geographic populations have now been identified with genetically inherited forms of amyloid. One group is found in Israel, predominantly among Sephardic Jews, and this disorder is called Familial Mediterranean Fever and it is characterized by amyloid deposition, along with recurrent inflammation and high fever. Another form of inherited amyloid is known as Familial Amyloidotic Polyneuropathy, and it has been found in at least three nationalities, namely, Swedish, Portuguese and Japanese. Amyloid deposition in this disease occurs predominantly in the peripheral and autonomic nerves. Hereditary amyloid angiopathy of Icelandic origin is a autosomal dominant form of amyloid deposition primarily affecting the vessels in the brain, and has been identified in 128 members distributed in at least 8 families restricted to a small geographic area of western Iceland. These patients clinically have massive cerebral hemorrhages in early life which usually causes death before the age of 40.
The primary, secondary and familial forms of amyloid that I have so far described tend to involve many organs of the body including heart, kidney, liver, spleen, GI tract and skin.
Isolated forms of amyloid, on the other hand, tend to involve a single organ system. Isolated amyloid deposits have been found in the lung, and heart. Up to 90% of type II diabetic patients (non-insulin form of diabetes) have isolated amyloid deposits in the pancreas restricted to the beta cells in the islets of Langerhans. Isolated forms of amyloid have also been found in endocrine tumors which secrete polypeptide hormones such as in medullary carcinoma of the thyroid. A serious complication of long-term hemodialysis is amyloid deposited in the medial nerve and clinically associated with carpal tunnel syndrome. By far the most common type of organ-specific amyloid, and amyloid in general, is that found in the brains of patients with Alzheimer""s disease. In this disorder, amyloid is predominantly restricted to the central nervous system (CNS). Similar deposition of amyloid in the brain occurs in Down""s syndrome patients once they reach the age of 35 years. Other types of CNS amyloid deposition include rare but highly infectious disorders such as Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome and scrapie in animals.
Current Classification of Amyloid: By Protein Type
It was misleading to group the various amyloidotic disorders strictly on the basis of their clinical features since, as shown in FIG. 34, when the major proteins involved were isolated and sequenced, they turned out to be different. For example, amyloid seen in rheumatoid arthritis and osteoarthritis, now known as AA amyloid, was the same amyloid protein identified in patients with the familial form of amyloid known as Familial Mediterranean Fever. Not to confuse this issue, it was decided that the best classification of amyloid should be according to the major protein type found, once it was isolated, sequenced and identified.
AA Amyloid
AA amyloid is common to a host of seemingly unrelated disorders including long-standing inflammation, various forms of malignancy, and in Familial Mediterranean Fever. Also it is the type of amyloid formed in animal models which use daily repeated injections of antigens. A potent antigen used today is casein or azocasein. In these animals, amyloid deposition occurs in the spleen, liver and kidney within 7-10 days of repeated injections.
The isolated amyloid protein in these cases turns out to be approximately 76 amino acids long, and having a MW of about 8,500. These 76 amino acids correspond to the amino terminal ⅔s of a naturally occurring serum protein known as SAA (Serum amyloid A). SAA is known to be an acute phase protein whose concentration increases about a thousand-fold, usually within 24 hours, during any inflammatory disorder. It is mainly made by the liver but current research also suggests that it can be found in other tissues as well. It""s normal function at this time is not really known. The AA amyloid protein that is deposited in the tissues is identical from patient to patient, regardless of the nature of the inflammatory disorder that has preceded its deposition. The AA amyloid protein is essentially the same protein found in many other species including mice, ducks, mink and monkeys. This conservation is probably due to the important, yet unknown, role that the precursor (SAA) plays during the process of inflammation.
AL Amyloid
Another major type of amyloid protein that has been identified is known as AL amyloid usually due to the deposition of the variable region of immunoglobulin light chains, either lambda or kappa chains; but it may also consist of the entire light chain. Since AL amyloid represents the variable region of light chains, AL amyloid isolated from different patients differs in its amino acid sequence. However, within a single patient the sequence of the AL amyloid protein is constant regardless of the organ from which the amyloid is isolated. This amyloid usually occurs secondary to multiple myeloma, or B-cell type malignancies (ex. immunoblastic lymphomas) and other plasma cell dyscrasias. Not all patients with multiple myeloma develop AL amyloid. Only 10-15% develop clinical problems related to these amyloid deposits, whereas a larger percent may have only microscopic deposits. Why all patients with immunoglobulin secreting abnormalities do not develop this type of amyloid is not known.
Prealbumin or Transthyretin
Another type of amyloid protein that has been identified is known as prealbumin or transthyretin. Prealbumin refers not to the precursor of circulating albumin, but to the serum protein that, in standard electrophoretic separation, migrates ahead of albumin. Prealbumin or transthyretin is normally the serum carrier of thyroxine, as well as retinol binding protein and retinoic acid. It has a normal plasma concentration of 20-40 mg/dL and is synthesized by the liver, and consists of 127 amino acids. A variant protein has been found in most types of Familial Amyloidotic Polyneuropathy (FAP) both the normal and the abnormal forms of transthyretin are found in the deposits, but the latter tend to predominate. Single amino acid substitutions have been identified at positions 30, 33, 60, 77, 84, 111 and 122 of the prealbumin molecule in the circulating plasma and in amyloid deposits of FAP patients. The most common substitution is a methionine for valine at position 30 of the prealbumin molecule. It is not clear how these amino acid substitutions allow to change the metabolic characteristics of transthyretin such that it is deposited primarily in peripheral and autonomic nerves. Immunological, chemical and direct DNA tests have been developed for detection of the specific mutations in the prealbumin variants. These assays are useful in identification of family members at risk while still in the preclinical phase of the disease.
A similar prealbumin molecule is deposited in the heart in an isolated form of amyloid known as senile cardiac amyloid. This type of amyloid is a frequent postmortem finding in patients more than 80 years of age and has been generally regarded as nonspecific.
Beta2-microglobulin
Another recent type of amyloid protein which has been identified is known as beta2-microglobulin. This type of amyloid has become recognized as a serious complication of long-term hemodialysis. Its most prominent clinical presentation is the carpal tunnel syndrome. Other manifestations include joint swelling, multiple spontaneous fractures and radiolucency in the wrist and hip. Its incidence correlates mainly with the number of years spent on dialysis, usually up to 50% of patients on hemodialysis over 8-10 years develop this type of amyloid. Beta2-microglobulin is a single polypeptide chain of 100 amino acid residues and has a MW of about 11,800. Beta2-microglobulin accumulates not only in the blood of uremic patients but also in the synovial fluid and in the tissues. The pathogenesis of dialysis amyloidosis is poorly understood. Some investigators have speculated that dialysis membrane characteristics may play a role, but this is controversial.
Procalcitonin
Several forms of isolated amyloid associated with endocrine tumors have been recently described. Where there are data characterizing the protein, the amyloid is derived from a portion of the normal hormonal product secreted (or prehormone synthesized) by the cells from which the tumor arises. Medullary carcinoma of the thyroid is such an example. The tumor is related to the C-type cells of the thyroid, which normally secrete calcitonin. Immunological studies have demonstrated that the amyloid in these tumors is a fragment of procalcitonin. A variant of calcitonin has been identified as the amyloid seen in the Islets of Langerhans in patients with diabetes. Atrial natriuretic factor, or a portion thereof, is deposited in isolated atrial amyloid.
Beta Amyloid Protein or xcex2/A4
Clinically the most common form of amyloidosis, is the type that is deposited in the brains of patients with Alzheimer""s disease, as well as in Down""s syndrome patients, usually over the age of 35. The amyloid protein deposited is now known as the beta-amyloid protein or xcex2/A4 (due to its known MW of approximately 4,200). It is derived from a larger precursor molecule known as the beta-amyloid precursor protein. This latter protein may take on different forms, including proteins of 695, 714, 751 and 770 amino acids, since the BAPP gene produces at least four principal mRNAs through alternative splicing of two exons.
PrP Protein
The last type of amyloid protein which I will discuss, which has been identified is known as the PrP protein or PrP 27-30, due to its having a MW of 27,000-30,000. This protein is also known as the prion protein PrP 27-30 was found to be derived from a larger protein, known as PrP Sc.
These proteins are highly infectious and are transmissible. They are found in the amyloid deposits in rare neurological disease such as Creutzfeldt-Jakob Disease, Gerstmann Strausller Syndrome; and kuru.
These diseases are usually rapidly progressive neurological disorders characterized by dementia, and fall into the category of subacute spongiform encephalopathies. Microscopically, the cerebral tissue is characterized by neuronal loss, gliosis, spongiforrn changes and extracellular amyloid deposits in the form of plaques. This type of amyloid deposition is also seen in sheep and hamsters and is known as scrapie. Recent evidence suggests that a small nucleic acid may be present, therefore, it may be regarded as a viral form of amyloidosis.
General Pathogenetic Mechanisms in Amyloidosis
Although amyloid deposits in all clinical conditions share common physical properties relating to the presence of a beta-pleated sheet conformation, it is now clear that many different chemical types exist and additional ones are likely to be described in the future. You may see that there are several common pathogenetic mechanisms that may be operating in amyloidosis in general. In most cases, a circulating precursor protein may result from overproduction of either intact or aberrant molecules (plasma cell dyscrasias), reduced degradation or excretion (SAA in some secondary amyloid syndromes and beta2-microglobulin in long-term hemodialysis), or genetic abnormalities associated with variant proteins (FAP). Proteolysis of a larger protein precursor molecule occurs in many types of amyloidosis, resulting in the production of lower MW fragments that polymerize and assume a beta-pleated sheet conformation as tissue deposits, usually in an extracellular location. What are the precise mechanisms involved, and the aberrant causes leading to changes in proteolytic processing and/or translational modifications is not known in most amyloids.
Proteoglycans and Glycosaminoglycans in Amyloidosis: a Specific Component of All Amyloids (FIG. 35)
The presence of PGs and/or GAGs associated with amyloid has been known for some time. Virchow (1854) first suggested the presence of carbohydrate in amyloid deposits, when he demonstrated positive iodine staining in organs infiltrated with amyloid indicating the presence of starch or cellulose. This material was termed xe2x80x9camyloidxe2x80x9d meaning xe2x80x9cstarch-likexe2x80x9d. Friedrich and Kekule (1859) later demonstrated the protein nature of these deposits.
It was not until the late 1960s and early 1970s that the true nature of the carbohydrate present in amyloid deposits was determined. Increased amounts of GAGs (referred to as xe2x80x9cacid mucopolysaccharidesxe2x80x9d) were demonstrated in tissue from human autopsies which were infiltrated with amyloid (Berenson et al., 1969; Bitter and Muir, 1965; 1966; Dalferes et al., 1967; 1968; Mowry and Scott, 1967; Okuyara and Turumi, 1963; Pennocket et al., 1968; see the appended Citations). Acid mucopolysaccharides were also described in the experimental induction of amyloidosis (Dalferes et al., 1968). Usually highly sulfated GAGs, such as heparan sulfate were increased in the amyloidotic liver and spleen of the AA and AL forms of amyloid (Bitter and Muir, 1966; Okuyara and Turumi, 1963; Pennock et al., 1968) whereas in cardiac tissue infiltrated with amyloid (i.e. senile cardiac amyloid) hyaluronic acid was shown to be the main GAG (Berenson et al., 1969). Since these studies only assessed the GAG composition in amyloid deposits obtained at end stages of the disease process (at autopsy), there was no indication of whether GAG accumulation was a primary, secondary or concurrent process with amyloid deposition. In addition the precise location of GAG accumulation in relation to amyloid deposition was also not known.
In 1985, Snow and Kisilevsky demonstrated that both highly sulfated GAGs and the amyloid protein were deposited in tissues (spleen, liver and kidney) at virtually the same time and in the exact same location using an experimental model of AA or inflammation-associated amyloidosis. In two different models of amyloid induction, neither the nature of the inflammatory inducing agent nor the length of time of the inflammatory reaction influenced the concurrent deposition of amyloid protein and GAGs. This initial study suggested that highly sulfated GAGs such as heparan sulfate, heparin and/or keratan sulfate were involved. These results indicated that the accumulation of GAGs was not a general reaction to an inflammatory stimulus but was specifically related to amyloid deposition itself. A subsequent study (Snow et al., 1987) confirmed that heparan sulfate and/or heparin were the only GAGs accumulating in the spleen in association with the AA amyloid protein. In this latter investigation plasma GAGs were found to be elevated (2-3 fold) at the time of GAG deposition in the tissues (spleen, liver). However, since most of this increase was due to chondroitin-4-sulfate, it suggested that the heparan sulfate/heparin increase observed in the tissues was probably derived from the accumulation of GAGs synthesized at the sites of amyloid deposition. Experiments using radioactive precursors for GAG synthesis demonstrated that a significant increase in GAG synthesis occurs in amyloidotic tissue in comparison to controls (Snow, unpublished data).
Although the sites of GAG synthesis in various systemic organs during amyloid deposition has not yet been identified, light microscopic (Snow et al., 1987; Snow et al., 1988) and ultrastructural (Snow et al., 1991) studies suggest that endothelial cells and/or reticuloendothelial cells may be involved. In fact, the initial accumulation of GAGs and amyloid in experimental AA amyloidosis occurs in the perifollicular sinusoids of the spleen and the walls of the central veins in the liver, two sites having a predominance of reticuloendothelial and/or endothelial cells.
Some of the first clear evidence for an intimate relationship between AA amyloid and highly sulfated PGs (such as heparan sulfate) are derived from an ultrastructural study which demonstrates positive Ruthenium red and Cuprolinic blue staining associated with amyloid fibrils in both amyloidotic spleen and liver, as well as in isolated fibril preparations (Snow et al., 1987). These cationic dyes stain sites enriched in PGs.
Recent immunocytochemical evidence confirms the histochemical results and demonstrates that heparan sulfate is specifically localized to sites of amyloid deposition in the mouse model of AA amyloidosis (Snow et al., 1991). Both an affinity-purified polyclonal antibody and a monoclonal antibody (HK-102), each recognizing specific domains on the protein core of a basement-membrane derived heparan sulfate proteoglycan (HSPG) localized HSPG core protein to the sites of amyloid deposition in both spleen and liver in experimental AA amyloidosis. In addition, a monoclonal antibody (HK-249) directed against the GAG chains of the basement-membrane derived HSPG demonstrated that the GAG chains were also localized to these same areas of amyloid deposition (Snow, Kisilevsky, Kimata, Kato and Wight, unpublished data). Furthermore, immunogold labelling at the ultrastructural level (Snow et al., 1991) demonstrated that the protein core of the HSPG was localized primarily to the amyloid fibrils which accumulate in the spleen and liver. These latter studies suggest that the heparan sulfate accumulating in the tissue concurrent with the AA amyloid protein involves deposition of both the protein core and GAG chains and is likely in the form of a HSPG proteoglycan.
The presence of highly sulfated GAGs in amyloidotic tissue, the close temporal relationship between initial amyloid deposition and PG accumulation in experimental AA amyloidosis, and the intimate ultrastructural association between AA amyloid fibrils and heparan sulfate proteoglycans, all imply that PGs may play a role in the pathogenesis of AA amyloidosis and that this role may be common to all types of amyloid. If this were true, then we would expect highly sulfated GAGs and/or PGs to be present in all types of amyloid regardless of protein type. Studies involving histochemical staining techniques employing the Alcian blue-magnesium chloride staining technique, as well as biochemical analysis, and immunocytochemical studies were used to test this hypothesis. The types of amyloid analyzed are shown in Table 1B. The results demonstrated that sulfated GAGs and/or PGs are present in close association with all these amyloids regardless of the nature of the amyloid protein deposited, the tissue or organ involved, or the extent of deposition.
Preliminary analysis involving immunohistochemical techniques using a variety of antibodies to different PG and GAG epitopes suggests that heparan sulfate proteoglycans are present in human AA and AL amyloid deposits in a number of different organs within the same individual (Snow, Benditt, Kimata, Kato, Hassell and Wight, unpublished data) suggesting that this particular highly sulfated proteoglycan may be involved in amyloid deposition independent of the nature of the amyloid protein involved and the type of organ or tissue in which the deposition occurs.
Possible Role(s) of Proteoglycans in the Pathogenesis of Amyloidosis
One possible general role that the highly anionic PGs such as heparan sulfate may play in amyloidosis, is to influence different amyloidogenic proteins to form similar beta-pleated sheet structures and demonstrate similar morphological (i.e. fibrillar structure), staining (Congo red and Thioflavin S positive) and spectral characteristics. The ability of PGs/GAGs to influence amyloid fibril formation can be analogous to its role in collagen fibrillogenesis where the presence of PGs/GAGs in the early stages of collagen fibril formation determined the rate and the size of the fibril formed (Gelman and Blackwell, 1974a; 1974b; 1974c). Further studies are needed to determine which GAGs and/or PGs (if any) influence the conformation of other amyloidogenic proteins such as the beta-amyloid precursor in Alzheimer""s disease, the PrP protein in the prion diseases, or the immunoglobulin light chains in AL amyloid.
Although it is feasible that highly sulfated PGs may play a role in the conformational folding of precursor amyloidogenic proteins into amyloid fibrils, many investigators would argue that PGs have nothing to do with amyloid fibril formation. This is due to the fact that a number of studies have shown that intact amyloid proteins (i.e. beta2-microglobulin) and/or synthetic peptides to portions of amyloid proteins (i.e. A4) can adopt a beta-pleated sheet conformation and polymerize as fibrils in vitro (Conners et al., 1985; Glenner et al., 1971). These studies include the formation of amyloid-like fibrils in vitro from immunoglobulin light chains (AL amyloid) (Glenner et al., 1971), beta2-microglobulin (Conners et al., 1985) and synthetic peptides corresponding to portions of the A4 amyloid protein (Castano et al., 1986; Gorevic et al., 1987; Kirschner et al., 1987). However, diverse conditions and treatments appeared necessary for amyloid fibril formation to occur, including a progressive decrease in salt concentration for beta2-microglobulin (Conners et al., 1985) and enzymatic digestion in an acidic environment for light chains of immunoglobulins (Glenner et al., 1971). In addition, in some instances, properties of the formed synthetic amyloid fibrils were somewhat different that those found in vivo. For example, the synthetic amyloid fibrils formed from synthetic peptides to various regions of the A4 protein could be completely solubilized whereas isolated amyloid fibrils from brain tissue is known to be very insoluble and tends to aggregate. Further studies are needed to determine whether the type of self-aggregation which can occur in vitro under a number of different conditions with various amyloidogenic proteins and synthetic analogues is actually the mechanism operating in vivo at the sites of amyloid formation.
A second possible role of highly sulfated proteoglycans in amyloidosis may be in determining the anatomical location of amyloid deposition. During the experimental induction of amyloid (i.e. AA amyloidosis, amyloid plaque formation in the scrapie hamster model), it is not known why the deposition of amyloid always occurs in precise anatomical sites (i.e. perifollicular area of spleen and in walls of central veins in liver; subependymal localization in scrapie hamster model). Likewise, clinically, certain types of amyloid are always found in specific organs or locales as opposed to other types of amyloid. Examples include senile cardiac amyloid in the heart, amyloid plaques in the hippocampal region in Alzheimer""s disease and amyloid confined to the peripheral and autonomic nervous system in familial amyloidotic polyneuropathy. If highly sulfated PGs and/or GAGs are important in determining the location of amyloid deposits then it is possible that the PGs and/or GAGs are present in tissue locations prior to the deposition of amyloid to these sites. Although it is difficult to determine whether the association of PGs/GAGs to areas of amyloid deposits occurs as a primary or secondary event, evidence to suggest that they are deposited early during amyloid formation comes from a number of studies. In experimental AA amyloidosis, time course studies have demonstrated that highly sulfated GAGs (now identified as HSPGs) and the AA amyloid protein are deposited at virtually the same time and in the same locale (Snow and Kisilevsky, 1985; Snow et al., 1991). In Alzheimer""s disease, immunocytochemical studies have demonstrated that HSPGs are present in xe2x80x9cprimitive plaquesxe2x80x9d (Snow et al., 1988). These plaques, containing essentially no amyloid component, are believed to be the precursor form of the mature plaque which contains an abundance of amyloid localized to a central core. Time-sequence analysis of initial xcex2/A4 and HSPG deposition in different aged patient""s with Down""s syndrome indicate that HSPG accumulation in association with xcex2/A4 may occur prior to the formation of fibrillar amyloid (Snow et al., 1990a), suggesting an important early role for HSPGs in the pathogenesis of xcex2/A4 formation and/or deposition.
Another possibility that may be important in determining the location of amyloid deposits is that PGs have a strong binding affinity with possible amyloidogenic precursor proteins and/or the amyloid proteins themselves. As previously discussed, HSPGs appear to have a strong binding affinity for both the AA amyloid protein and the xcex2/A4 amyloid protein (Snow et al., 1989) which may be important in the ultimate deposition of these amyloidogenic proteins to specific sites containing increased amounts of HSPGs. The preferential binding of PGs to specific proteins is not a new concept. For example, it has been known for some time that particular PGs interact with the protein moiety of beta-lipoproteins (Camejo, 1982).
A third possible role of highly sulfated PGs in the pathogenesis of amyloidosis, regardless of whether the accumulation of PGs in amyloid deposits occurs as a primary or secondary event, may involve PGs contributing to the insolubility of amyloid and its inaccessibility to proteolytic degradation in tissues. Amyloid deposits once established in various organs and tissues are very stable and usually stay in these sites over long periods of time without significant change (Franklin, 1972; Glenner and Page, 1976). Both experimentally and clinically, few methods have been successful for the removal of amyloid deposits once formed. In this same context, the association of PGs and/or GAGs with the amyloid protein in the neuritic plaques and the components of neurofibrillary tangles in Alzheimer""s disease may contribute to the insolubility problems investigators have had in trying to isolate and sequence amyloid proteins (Gorevic et al., 1986; Iqbal et al., 1984). The presence of PGs at the sites of amyloid deposition may also be involved by interacting with proteases and/or protease inhibitors (i.e. alpha1-antichymotrypsin) (Abraham et al., 1988) which may lead to the prevention of amyloid degradation once formed. An analogous situation has been recently reported (Saksela et al., 1988) in which endothelial cell-derived heparan sulfate was found to protect basic fibroblast growth factor from proteolytic degradation.
It is interesting to speculate that in Alzheimer""s disease specific alterations in PG metabolism (i.e. synthesis and/or degradation) may occur in the brains of patients afflicted with this disease. It is even possible that the primary defect in Alzheimer""s disease may actually be a defect directly or indirectly affecting PG metabolism which is only apparent in some individuals with aging and/or the presence of an unknown environmental stimulus. This PG defect could be residing in a subpopulation of cells and may contribute to the accumulation of PGs only within specific areas of the brain (i.e. in pyramidal neurons in the hippocampus). This may help explain the xe2x80x9cselective vulnerabilityxe2x80x9d of neurons that are affected in Alzheimer""s disease. It is important to note that changes in the sulfation and/or polymerization of neuronal GAGs may occur in the brain as a function of aging. This may be necessary for the formation and/or accumulation of plaques and/or tangles that are observed even in aged non-demented individuals.
Alzheimer""s Disease, Amyloid Precursor Proteins and the xcex2/A4 Peptide
The characteristic and diagnostic feature of brains of individuals with AD is the deposition and accumulation of a 39-43 amino acid peptide (Mrxcx9c4,200) termed the beta-amyloid protein (Glenner and Wong, 1984; Wong et al., 1985), A4 (Masters et al., 1985) or xcex2/A4 (Beyreuther and Masters, 1990). This small peptide is a major component within the amyloid deposits of neuritic plaques and in the walls of blood vessels (i.e. congophilic angiopathy) in the brains of patients with AD, and is derived from larger precursor molecules (termed beta-amyloid precursor protein or APP) by an unknown pathogenic mechanism. APP is composed of 3 major isoforms, APP695, APP751, and APP770, which arise by alternative splicing of a single gene (Kang et al., 1987; Kitaguchi et al., 1988; Ponte et al., 1988). APP751 and APP770 contain a protease inhibitor domain (Ponte et al., 1988; Kitaguchi et al., 1988). The normal function of the APP is not known but some have speculated that it may play a role in neuronal adhesion (Kang et al., 1987), and/or neuronal growth and regulation (Whitson et al., 1989; Saitoh et al., 1989). APP contains an extracellular amino-terminal domain, a single transmembrane domain, and a short cytoplasmic segment. The sequence of xcex2/A4 includes the first 28 residues of the extracellular domain and 11-14 residues of the proposed transmembrane domain. Recent studies (Sisodia et al., 1990; Esch et al., 1990) indicate that during normal APP catabolism the intact amyloidogenic fragment containing the xcex2/A4 is not generated, due to proteolytic cleavage at or near position 612 of APP (Lys-16 of xcex2/A4), resulting in the release of a soluble secreted protein of undefined function. These latter studies also suggest that altered APP processing is required to release the xcex2/A4 domain found as a major component of fibrillar amyloid deposits.
Proteoglycans and Glycosaminoglycans
Proteoglycans are a group of complex macromolecules which are found in all organs and tissues, intracellularly in a variety of different cell types, or extracellularly in the matrix where they are exported for a variety of functions (Gallagher et al., 1986; Hascall and Hascall, 1981; Poole, 1986; Ruoslahti, 1988). Proteoglycans consist of a protein core to which one or more GAG chains are covalently linked through o-glycosidic linkage to serine residues in the core protein (Hascall and Hascall, 1981; Hassell et al., 1986). The highly anionic GAG chains consist of repeating disaccharide units, containing 1) hexosamine (either D-glucosamine or D-galactosamine) and 2) hexuronic acid (either D-glucuronic acid or L-iduronic acid) (Muir, 1969). Specific disaccharide repeat patterns give rise to 7 different types of GAGs. These are hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate. Usually one type of GAG predominates on a single core protein, giving rise to four major families including chondroitin sulfate proteoglycan (CSPG), dermatan sulfate proteoglycan (DSPG), heparan sulfate proteoglycan (HSPG) and keratan sulfate proteoglycan (KSPG). However, more than one type of GAG can be inserted on the same core protein giving rise to xe2x80x9chybrid PGsxe2x80x9d. The core proteins of the PGs have been the least well studied. However, a number of cDNAs have been cloned and the partial or complete amino acid sequence of some of these have been deduced (Bourdon et al., 1985; Day et al., 1987; Doegle, 1986; 1987; Krusius et al., 1987) including the complete amino acid sequence for the BM-HSPG (known as xe2x80x9cperlecanxe2x80x9d) in both mouse (Noonan et al., 1988; 1991) and human (Kallunki and Tryggvason, 1992; Murdoch et al., 1992).
The diversity of PGs largely derives from the number of different protein cores within each PG family and from the polydiversity produced by a number of post translational modifications required to construct the final molecules. This structural diversity may account for the multiple properties of these complex molecules.
Specific Proteoglycans in Amyloidogenesis
Specific PGs such as the HSPG, together with the amyloid P component (Coria et al., 1988; Holck et al., 1979) are the only macromolecules consistently associated with all varieties of amyloid irrespective of the type of amyloid protein present or its location (Snow and Wight, 1989b). It is likely that some components involved in amyloid formation, deposition and accumulation are not unique to only one type of amyloidosis (i.e. the xcex2/A4 of AD), but play a role in the pathogenesis of other types of amyloid as well (i.e. AA or inflammation-associated amyloid, AL amyloid involving the deposition of immunoglobulin light chains; transthyretin in familial amyloidotic polyneuropathy). A common mechanism may help explain why different types of amyloid (i.e. consisting of different amyloid proteins) all form similar beta-pleated sheet structures with similar morphological (i.e. fibrillar structure), staining (Congo red and Thioflavin S positive), and spectral characteristics.
One general role that the highly anionic PGs may play in amyloidosis is to influence different amyloidogenic proteins to form similar beta-pleated sheet structures. The ability of PGs and GAGs to influence amyloid fibril formation might be analogous to their role in collagen fibrillogenesis where the presence of PGs and GAGs determines the rate and the size of the fibril formed (Katz et al., 1986; Mathews and Decker, 1968; Obrink, 1973; Vogel et al., 1984). Evidence that highly sulfated GAGs can influence protein folding comes from in vitro circular dichroism studies which demonstrated that different GAGs can exert a direct influence on the conformational folding of various mixed and homologous polypeptides (Gelmann and Blackwell, 1973; 1974). Recent studies (McCubbin et al., 1988) demonstrate that heparan sulfate (and not heparin or chondroitin-6-sulfate) influences the specific AA amyloid precursor, known as SAA2 to form a predominant beta-pleated sheet structure. This study implicates heparan sulfate in conformational alterations of amyloidogenic precursors which may be important in the ultimate formation of the beta-pleated sheet structure characteristic of all amyloids.
The distribution of particular proteoglycans may also specifically direct the anatomical location of amyloid deposition. If so, then PGs and/or GAGs may be present in tissue locations prior to the deposition of fibrillar amyloid at these sites. Although it is difficult to establish definitively whether the association of PGs/GAGs with amyloid deposits occurs as a primary or secondary event, evidence to suggest that PGs/GAGs are deposited early or concurrent with amyloid formation comes from a number of studies (Snow et al., 1988b; Snow et al., 1990). For example, in experimental AA amyloidosis, time course studies have demonstrated that HSPGs and the AA amyloid protein are deposited at virtually the same time and in the same locale (Snow and Kisilevsky, 1985; Snow et al., 1988b).
Regardless of whether the accumulation of PGs in amyloid deposits occurs as a primary or secondary event, PGs may contribute to the stability of amyloid and its inaccessibility to removal or proteolytic degradation in tissues. The presence of PGs at the sites of amyloid deposition may inactivate proteases and/or activate protease inhibitors (i.e. alpha 1-antichymotrypsin) (Abraham et al., 1988), which may prevent amyloid degradation. Recent studies have demonstrated that endothelial cell-derived heparan sulfate protects basic fibroblast growth factor from proteolytic degradation (Saksela et al., 1988). It is postulated that the basement membrane derived HSPG once bound to xcex2/A4 prevents its normal degradation by proteases.
Heparan Sulfate Proteoglycans and Their Potential Role in xcex2/A4 Amyloidogenesis
Although there are potentially many different types of PGs and/or GAGs that can be synthesized by cells, both within and outside the CNS, accumulating evidence suggests that only the HSPG class is localized to a variety of different amyloids (Snow et al., 1988a; 1988b; 1989). The specific association of HSPGs with a variety of different amyloids suggests that the HSPG-amyloid association is more than simply a charge effect since similarly charged PGs such as keratan sulfate do not appear to be present in association with different amyloid proteins (Snow and Wight, 1989). Immunocytochemical evidence indicates that it is the basement membrane derived HSPG (i.e. Perlecan) which is associated with a variety of different amyloids (Snow et al., 1988a; 1988b; Snow and Wight, 1989). The deduced amino acid sequence of the core protein of this particular PG is known from the analysis of corresponding cDNA sequences in both mouse (Noonan et al., 1988; 1991) and human (Kallunki and Tryggvason, 1992; Murdoch et al., 1992), and the gene for this HSPG is localized to chromosome 1 in both of these species (Wintle et al., 1990).
As described above, recent studies (Esch et al., 1990; Sisodia et al., 1990) imply that other amyloid components may be necessary and involved in post-translational modifications of the APP, ultimately leading to the accumulation of the xcex2/A4 fragment. One such component may be the basement membrane derived HSPG. Our previous studies have shown the accumulation and co-localization of a specific HSPG to the amyloid deposits containing xcex2/A4 in neuritic plaques and amyloidotic blood vessels in AD and Down""s syndrome brain (Snow et al., 1988a; 1989; 1990). Immunohistochemical (Snow et al., 1988; Snow and Wight, 1989) and cationic dye studies (Snow et al., 1989; Young et al., 1989) indicate an intimate ultrastructural association between this particular PG and amyloid fibrils containing the xcex2/A4.
In Down""s syndrome, evaluation of brains from patients aged 1 day to 51 years demonstrated initial co-accumulation of both HSPG and xcex2/A4 in diffuse cortical deposits in 18 and 24 year old patients. These deposits were congo red and thioflavin S negative and were believed to be the precursor to fibrillar amyloid formation which was observed in Down""s syndrome patients over the age of 35 years (Snow et al., 1990). This observation rules against the possibility that PGs accumulate in amyloid simply as a common response to the presence of amyloid fibrillar deposits.
Potential Animal Models of Alzheimer""s Disease
Early animal models of AD focused upon the neurochemical and anatomic pathology of the disease and attempted to mimic this pathology with lesions in animals. One of the first models focused on the cortical cholinergic system which is profoundly affected in AD (Perry et al., 1977; Davies and Maloney, 1978). Whitehouse et al. (1982) reported that the cells of origin of the cortical cholinergic fibers, located in the nucleus basalis, were substantially depleted in AD. This led to a large number of studies examining the effects of nucleus basalis lesions in rats and other species on behavioral and neurochemical indices. These lesions were widely reported to induce memory deficits for a number of tasks (Flicker et al., 1983; Helper et al., 1985; Kesner et al., 1986). Some normally aged rats have spontaneous loss of nucleus basalis cholinergic neurons, and this loss is correlated with memory dysfunction (Bartus et al., 1982). Fisher et al. (1987) found that intraventricular nerve growth factor infusions could reverse the cholinergic neuron atrophy and restore memory function in these aged rats. This finding, among others, has led to the suggestions that nerve growth factor (NGF) may be therapeutically beneficial in AD (Hefti et al., 1989). Arendash et al. (1987) reported that the long term effects (14 months postlesion) of nucleus basalis lesions caused the formation of plaque-like and fibrillar structures in rat brain, and resulted in neuron loss in cortex, hippocampus and amygdala. Surprisingly, additional reports characterizing this model of AD have not appeared over the last 5 years.
In spite of the Andendash et al. (1987) report, most view the cholinergic deficits as a critical component, but not the major dysfunction in AD. Some researchers have raised doubts about the role of basal forebrain cholinergic neurons in memory dysfunction (Wenk et al., 1986). Others suggest that the nucleus basalis neuron atrophy in AD may be secondary to cortical degeneration, because cortical lesions in rats and monkeys can lead to shrinkage of these cells (Sofroniew et al., 1983; Pearson et al., 1983). A second lesion model receiving some attention is lesions in entorhinal cortex. Hyman et al. (1984) recognized that loss of projection neurons from the entorhinal cortex to the hippocampus was a prominent feature in AD, which, together, with the subicular neuron loss, functionally isolated the hippocampus from the rest of the brain. Entorhinal lesions in rats have long been studied as a model system for synaptic sprouting within the denervated hippocampus. Geddes et al. (1986) have reported remarkable similarities in the hippocampus between neurochemical and anatomical changes in AD and those following entorhinal lesions in rats, and suggest that reactive synaptogenesis may play a critical role in amyloid plaque formation. However, one problem with most lesion models of AD is that they ultimately may mimic some of the end stage pathology of the disease, and not the pathogenesis of the disease.
Another potential animal model of AD is normal aging. Several species develop xcex2/A4 immunoreactive amyloid plaques including dogs (Ishihara et al., 1991), non-human primates (Selkoe et al., 1987; Martin et al., 1991), and polar bears (Cork et al., 1988). One problem with these models is that the amyloid plaques usually occur in late life in these animals, precluding rapid screening of potentially therapeutic substances or interventions. One advantage of the rat model described in this patent application is the rapid production (within 1 week) of xcex2/A4 amyloid deposits in brain. Interestingly, Vaughan and Peters (1981) reported that aged Sprague-Dawley rats (28 and 30 months old) possess congophilic amyloid plaques that ultrastructurally resemble the material found in AD. Surprisingly, these data have not been widely discussed in other reports.
More recently, transgenic mice have been produced as potential models of AD. The rationale for these models is that overproduction of an APP transgene (containing all or part of the APP sequence) could lead to the eventual development of xcex2/A4 deposits in mouse brain and subsequent plaque formation. Wirak et al. (1991) generated transgenic mouse lines containing the xcex2/A4 sequence under the control of the human APP promoter. After 1 year, these mice developed xcex2/A4 deposits within hippocampal neurons and formed aggregates of amyloid-like fibrils. Quon et al. (1991) used a full length APP751 (Kunitz protease inhibitor containing form) sequence linked to a neuron-specific enolase promoter. Transgenic mice with this construct displayed extracellular xcex2/A4 immunoreactive deposits, which were infrequently stained with thioflavin S, but not by congo red, suggesting a pre-amyloid like composition. Kawabata et al. (1991) developed transgenic mouse lines massively overexpressing a construct encoding the C-terminal 100 amino acids of APP under control of a Thy-1 element. These mice displayed pathology remarkably similar to that observed in AD including amyloid plaques, neurofibrillary tangles and neurodegeneration in hippocampus, neocortex and even cerebellum. While extremely promising, the report by Kawabata et al. (1991) has been retracted whereas the study by Wirak et al. (1991) has been questioned (Jucker et al., 1992). Additional research will determine if the overexpression of APP is sufficient to produce the neuropathological features of AD. While more rapid than aged animal models, these models still suffer from the requirement of several months delay before AD-like pathology may develop.
The last type of animal models all involve injecting or transplanting materials into brain parenchyma. Richards et al. (1991) grafted fetal hippocampus from trisomy 16 mice into congenic normal mice and observed intracellular staining for APP, xcex2/A4, neurofibrillary tangles, tau protein and ubiquitin immunoreactivity, within the graft, but not in host tissues. However, no extracellular xcex2/A4 deposits resembling amyloid plaques were reported. Trisomy 16 mice contain an additional APP gene, analogous to Down""s syndrome patients (human trisomy 21), who develop amyloid plaque and neurofibrillary tangle pathology similar to that observed in AD. A second model using parenchymal injections was described by Kowall et al. (1991). These authors acutely injected xcex2/A4 (1-40) into rat hippocampus and observed substantial neuronal loss and induction of ALZ-50 antibody immunostaining within 1 week. These effects were attenuated by coadministration of substance P either centrally or peripherally. Importantly, no apparent congophilic amyloid deposits was observed. A final animal model involved acute injections of purified AD amyloid plaque cores into rat cortex or hippocampus (Frautschy et al., 1991). At 1 month, but not 1 week, ALZ-50 and ubiquitin staining and neuronal degeneration was evident in 70% of the rats. As expected, these amyloid cores isolated from AD brain remained congophilic after residing in rat brain for 1 month. It is important to note that SDS-extracted amyloid cores from human AD brain still contain immunoreactivity for HSPGs (Snow et al.,) suggesting that in this latter study, besides undefined components in the isolated plaque cores, HSPGs and xcex2/A4 were present.
Each of the animal models described above possess specific features making them valuable for AD research. However, each has some disadvantages. The specific advantages of our model include 1) the deposition of congophilic xcex2/A4 amyloid in rat brain at selected sites, 2) the model is rapid with some features present as early as 1 week (although neurodegenerative effects may require longer incubations as in Frautschy et al. (1991)), 3) the injected materials consists of defined components enabling one to determine the effects of each component individually (by infusing them separately or in different concentration ratios), and 4) by continuous infusion we are able to deposit into brain larger quantities of material than by acute injections.
Excessive congophilic and fibrillar amyloid deposition, containing a 39-42 amino acid peptide termed xcex2/A4, is a characteristic and diagnostic feature observed in brains of patients with Alzheimer""s disease (AD). A rapid animal model of xcex2/A4 amyloid deposition in rat hippocampus is described after assessing continuous infusion of xcex2/A4 (1-40) in the presence or absence of specific proteoglycans/glycosaminoglycans. 100% of animals (6 of 6) receiving infusions for 1 week of synthetic xcex2/A4 (1-40) plus mouse perlecan, a basement membrane-derived heparan sulfate proteoglycan (HSPG), demonstrated congo red and thioflavin S deposits indicative of amyloid adjacent to the infusion site. Similar results (100%, 4 of 4) were obtained in animals infused for 2 weeks (and sacrificed 5 weeks later) with xcex2/A4 (1-40) plus mouse perlecan. Extracellular amyloid fibrils with a diameter of 10-12 nm were identified by electron microscopy and were specifically immunogold decorated with xcex2/A4 antibody. xcex2/A4 amyloid deposition also occurred in 4 of 6 animals infused with xcex2/A4 (1-40) only when endogenous brain HSPG accumulated at the xcex2/A4 infusion site.
Other new potential animal models of AA amyloidosis, AL amyloidosis, Prealbumin/Transthyretin amyloidosis, Beta2-microglobulin amyloidosis, Endocrine type amyloidosis, and PrP protein amyloidosis are presented involving injections and/or infusions of the various amyloid proteins in combination with perlecan or derivatives thereof. The disclosed animal models serve as in vivo screens for potential therapeutic intervention or agents in each of the different amloidoses.
In vitro studies demonstrate a particular high binding affinity between the high Mr basement membrane derived HSPG and xcex2/A4 (1-28) which could be competitively inhibited by highly sulfated GAGs such as heparin, heparan sulfate and related macromolecules (e.g., dextran sulfate). These in vitro studies indicate that highly sulfated GAGs are candidate agents for therapeutic intervention. This was confirmed by in vivo studies demonstrating that co-infusion of HS glycosaminoglycans with xcex2/A4 inhibited congophilic and fibrillar amyloid deposition and/or its persistence in brain. Additionally, since sequence analysis suggests that a putative heparin binding domain resides at residues 11-17 of xcex2/A4 (Cardin and Weintraub, 1989), a potential inhibitor of HSPG-xcex2/A4 binding and/or xcex2/A4 amyloid deposition in brain consists essentially of a small 6 amino acid peptide having the sequence of -valine-histidine-histidine-glutamine-lysine-leucine-.
The disclosed animal models will aid investigators in further determining the in vivo effects of xcex2/A4 amyloid on endogenous brain components, serve as rapid in vivo screens for candidate therapeutic interventions or agents, and further implicate highly sulfated macromolecules such as heparin, heparan sulfate, and related analogues, and a 6 amino acid peptide to the putative heparin binding domain of xcex2/A4, as inhibitors of xcex2/A4 amyloid deposition in brain and therefore as a potential therapeutic intervention for patients with AD and/or other amyloidoses.